Generation and propagation of the vectors
General cloning techniques were used to generate each of the vectors. Detailed protocols are available upon request. All the lentiviral Destination vectors are derived from the p156RRL-sinPPT-CMV-GFP-PRE/Nhe I vector 
and all the retroviral Destination vectors are derived from the pQCXI series (Clontech). For the lentiviral vectors, a linker was inserted between the Kpn I and Eco RI sites to clone the drug selection cassettes following the Woodchuck post-transcriptional response element (WPRE). All Destination vectors were propagated in the E.coli
strain DB3.1 (Invitrogen) under appropriate antibiotic selection (see plasmid maps for details). All Entry vectors were propagated in the E.coli
strain TOP10F' (Invitrogen) under kanamycin selection. After the LR recombination reaction, lentiviral vectors were propagated in the E.coli
strain Stbl3 (Invitrogen) and retroviral vectors in TOP10F', all under ampicillin selection, except for the pLenti CMV/TO Zeo DEST (zeocin).
cDNAs, shRNAs and miRNAs
The HA epitope-tagged Asf1a (NCBI GeneID: 25842) and Asf1b (GeneID: 55723) cDNAs as well as the HIRA (GeneID: 7290) cDNA were a gift from Peter Adams (Beatson Institute, Glasgow). The HA-Asf1a and HA-Asf1b were subcloned in the pENTR1A no ccdB plasmid and the HIRA cDNA was cloned into the pENTR4-V5 plasmid. The XPG (GeneID: 2073) cDNA cloned in to the pENTR3C vector with a V5 epitope and a GFP-fusion at its C-terminus (XPG-V5-GFP) was a gift of Ely Kwoh. The XPG-V5 was subcloned into pENTR3C to remove the GFP fusion. The Ubc9 (GeneID: 7329) cDNA was a gift from Claude Gazin (CNRS, UMR217). The ARID4B (GeneID: 51742) cDNA was obtained from the Kazusa cDNA project (clone HH11923, Accession #AB210032). The ARID4B cDNA was cloned in the pENTR4-V5 plasmid. A region of the cDNA encoding the shorter isoform was amplified by RT-PCR from U2OS cells and subcloned into the pCR2.1 TA cloning plasmid (Invitrogen) and sequence verified. The fragment was excised using the Bbv CI/Hind III restriction enzymes and inserted into the Bbv CI + Hind III-digested pENTR4-V5-ARID4B plasmid to generate pENTR4-V5 ARID4B Δchromo.
The shRNA for Asf1a was derived from an siRNA previously used 
. We inserted the following annealed oligonucleotides between the Bgl II/Hind III
sites of either pENTR/pTER+ or pENTR/pSUPER+:
5′GATCCCGTGAAGAATACGATCAAGTGTGTGCTGTCCACTTGATCGTATTCTTCACTTTTTGGAAA and 5′AGCTTTTCCAAAAAGTGAAGAATACGATCAAGTGGACAGCACACACTTGATCGTATTCTTCACGG, where the underlined sequence is specific for human Asf1a.
For MDC1: 5′GATCCCCCAACATGCAGAGATTGAAATTCAAGAGATTTCAATCTCTGCATGTTGTTTTTGGAAA and 5′AGCTTTTCCAAAAACAACATGCAGAGATTGAAATCTCTTGAATTTCAATCTCTGCATGTTGGGG
For XPG: 5′GATCCCAGAATACATGCGGTGGATTTTCAAGAGAAATCCACCGCATGTATTCTTTTTTGGAAA and AGCTTTTCCAAAAAAGAATACATGCGGTGGATTTCTCTTGAAAATCCACCGCATGTATTCTGG.
For the Asf1a shRNA, we also designed an miRNA-based loop in the shRNA because it was reported to result in better depletion efficiencies 
. To design the Asf1a miRNA, we used the algorithm from Open Biosystems (www.openbiosystems.com
). The following oligos were amplified with the Xho I and Eco RI amplification primers and subcloned into the pENTR/pSM2 vectors according to the manufacturer's protocol:
where the underlined sequences are specific for the human Asf1a.
LR recombination and purification of the plasmid DNA
The LR recombination was performed using the LR clonase mix (cat. #11791-019, Invitrogen) with 1 µl of miniprep DNA for each of the Entry and Destination vector, 4 µl of TE pH 8.0, 2 µl of LR buffer and 2 µl of LR clonase. Reactions were incubated at room temperature from 2 h to overnight. The proteinase K digestion step was omitted from the manufacturer's protocol and 2 µl of the reaction were used to transform 25 µl of competent E.coli Stbl3 or TOP10F' cells generated by the Zymo Research competent cell kit (cat #T-3002). Colonies were picked and inoculated in 50 ml of LB broth. 600 µl were used for a miniprep (Zippy, Zymo research) to confirm the identity of the clone and the remainder of the culture was used for a Qiagen Midi prep according to the manufacturer's instruction for transfecting 293T cells.
Transfection of 293T cells to generate third generation lentiviruses
The day before the transfection, 5×106 of 293T cells were seeded in a 10 cm dish. Transfection was done with 50 µl of Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions using 15 µg of the transfer vector, 15 µg of pLP1 (Invitrogen), 6 µg of pLP2 (Invitrogen), and 3 µg of pVSV-G (Invitrogen). The DNA:liposome complex (3 ml) was incubated with the cells in a final volume of 10 ml of OPTI-MEM (Invitrogen) overnight.
Transfection of 293T cells to generate second generation lentiviruses
The same procedure as above was used except that 20 µg of pCMVΔ8.9 
was used instead of the pLP1 and pLP2 vectors.
Transfection of 293gag/pol cells to generate retroviruses
The day before the transfection, 5×106
of 293 gag/pol cells were inoculated in a 10 cm dish. Lipofectamine 2000 was used for the transfection following the manufacturer's recommendations using 12 µg of the transfer vector, 1.2 µg of pGAG/POL 
and 1.2 µg of pVSV-G (Invitrogen).
Collection of viral supernatant and titration of viruses
At 48 h post-transfection, viral supernatants were collected and fresh OPTI-MEM (10 ml) was added to the dish for another collection at 72 h post-transfection. For each collection, viral supernatants were filtered through a 0.2 µm syringe filter. The 48 and 72 h collections were pooled, aliquoted and stored at −80°C. Viral titers were determined by seeding 6-well plates with 2.5×104 HT1080 or HeLa cells and transducing them with 10-fold dilutions (100-1 µl) of viral supernatant as described below. After 12–14 days, cells were fixed in cold methanol and stained with crystal violet solution (0.5% crystal violet, 25% methanol) and counted to determine their colony forming units (cfu). When necessary, viruses were concentrated in a Beckman SW28 rotor at 21,000 rpm for 2 h at 4°C and resuspended in 4 ml of Hank's Buffered Saline solution (HBS, Invitrogen). A second ultracentrifugation in a Beckman 55Ti rotor at 21,000 rpm for 90 minutes was performed and the viral pellet was resuspended in 100 µl of HBS and stored at −80°C in 10 µl aliquots. When measuring viral titers, we take into consideration several factors. First, the health of the 293T packaging cell is critical. If the cells are too confluent or not grown under optimal conditions, it will result in lower viral titers. Similarly, if the cells have been passaged for a long time, it will also yield lower titers. We always thaw a fresh vial of 293T cells when we are producing viruses. Second, the transfection procedure, liposome reagent, the quality of the media used as well as the amounts and quality of the vectors co-transfected are also critical. Third, the viral titers will also be proportional to the volume of media used to grow the cells in order to collect the viral supernatant. Finally, the cell line used for the titration, the amount of cells plated and the concentration of the selecting drug is also affecting viral titers. Other factors like the protein to be overexpressed or depleted might affect cell growth and viability and result in lower viral titers than expected. We recommend using a control vector expressing GFP to monitor transfection as well as transduction efficiencies.
Transduction of cell lines and induction with doxycycline
Cells were transduced at a MOI between 0.5 and 1. Viruses and cells were incubated overnight in D-MEM media (Invitrogen) containing 6 µg/ml of polybrene (Sigma) in a final volume of 950 µl for 6-well plates, 5 ml for 10 cm dishes and 11 ml for 15 cm dishes. The next day, the viruses were removed, the cells were rinsed twice with PBS and fresh media was added. For primary cells, a second round of transduction was done. Drug selection was added at 48 h post-transduction. The following concentrations were used: blasticidin: 2.5 µg/ml for WI38, HCA2, BJ cells and 5 µg/ml for the other cells, hygromycin: 100 µg/ml for WI38, HCA2, BJ cells, 200 µg/ml for U2OS cells and 300 µg/ml for HT1080 cells, neomycin: 300 µg/ml for WI38, HCA2, BJ cells and 800 µg/ml for the other cells, puromycin: 0.5 µg/ml for HeLa cells and 2.0 µg/ml for other cells, zeocin: 400 µg/ml for HT1080 cells and 200 µg/ml for other cells. Induction of the cDNA/shRNA/miRNA was typically done by addition of doxycycline at a final concentration of 1.0 µg/ml for 48 h (cDNA) or 96 h (shRNA/miRNA) unless indicated otherwise.
Generation of the MDC1 antibodies
The N-terminal 318 residues of MDC1 were expressed as a GST fusion in E.coli and separated from GST by cleavage with PreScission protease by glutathione sepharose (GE Healthcare) affinity. The domain was further purified by High Q (BioRad) ion exchange, and Superdex75 (GE Healthcare) size exclusion chromatography. Rabbits were immunized and antibodies were purified from sera by affinity chromatography. Both GST and GST-MDC1 N-terminus affinity columns were prepared by incubating glutathione sepharose with E. coli expression lysates overnight, washing the beads extensively with borate buffer, crosslinking the protein to the beads using dimethypimelimidate and ethanolamine, and washing with glycine-HCl and phosphate buffer. Sera was incubated with the GST affinity resin for 2 hours and then with the GST-MDC1 N-terminus affinity resin overnight. After extensive washes with PBS, antibody was eluted with 200 mM glycine-HCl pH 2.5 and immediately neutralized with 1 M K2HPO4. Antibody pools were dialyzed into PBS and stored at −80°C.
Other antibodies, protein electrophoresis, immunofluorescence
The following antibodies were used: anti-V5 (Invitrogen, R96025) at 1
1000 for Western blot and 1
1200 for immunofluorescence; anti-Asf1a (C6E10, cat#2990, Cell Signaling Technology) at 1
6000 for Western blot, anti-Asf1b (cat.#2769, Cell Signaling Technology) at 1
500 for Western blot, anti-HA (mouse monoclonal 12CA5) at a final concentration of 1 µg/ml for Western blot, anti-XPG 
, at 1
500 for Western blot, anti-p16 (Neomarkers, JC8) at 1
500 for immunofluorescence, anti-p21 (BD Biosciences, 556430) at 1
500 for immunofluorescence and anti-53BP1 (Bethyl Laboratories, BL182) at 1
2000 for immunofluorescence anti-GFP antibody (Abcam, Ab1218) at 1
1000 for Western blot, anti-GST (GE healthcare, 27-4577-01) at 1
100 for Western blot, anti-CBP (ICL Labs, RCBP-45A at 1
2000 for Western blot and 1
500 for immunofluorescence, anti-p150 (ss48, 
) at 1
1000 for Western blot, anti-PCNA clone PC10 
500 for immunofluorescence, α-tubulin hybridoma (DM1A, 
2000 for Western blot, anti-H3Ser10 clone 3H10 (Upstate, 05-806) at 1
2000 for Western blot, anti-c-myc 9E10 (Santa Cruz biotechnologies, sc-40) at 1
1000 for Western blot and 1
400 for immunofluorescence. Generation of cell extracts, protein electrophoresis, Western transfer and antibody detection were done using standard procedures. For immunofluorescence, cells were fixed in 4% paraformaldehyde in the case where GFP fluorescence was examined and 100% cold methanol in all other cases. Immunofluorescence was done as described before 
, and images were acquired on an Olympus BX60 fluorescence microscope with the spotfire 3.2.4 software (Diagnostics Instruments) and further processed with Photoshop CS2 (Adobe). For the MDC1 immunofluorescence, images were acquired on a Zeiss Axioskop using a Kodak DC120 digital camera and its capture software and further processed with Photoshop CS2. Images for the NTAP/PCNA, c-myc, V5 were collected on a Zeiss Axioplan 2 microscope with the Axiovision 4.5 software and processed with Photoshop CS3.
Live cell imaging
Bright field and fluorescent live cell pictures were taken on an Axiovert 200 microscope (Zeiss) using a Qicam fast camera (Qimaging) and the Openlab 4.0.4 software. The pictures were exported to Adobe Photoshop CS for brightness/contrast adjustments.
CAF-1 purification from HeLa S3 T-Rex-NTAP-p150 cells
Nuclear extracts were prepared from cells induced with 1 µg/mL doxycycline as described 
followed by a modified TAP method 
. Briefly, nuclear extracts (400 mM NaCl) were diluted two fold with Buffer A (25 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% NP-40, 10% glycerol, 1 mM DTT, 0.1 mM PMSF) to reduce the NaCl concentration to 200 mM final. Extracts were incubated with streptavidin sepharose (GE Healthcare) for three hours, rotating at 4°C. Beads were washed three times with large volumes of Buffer A+200 mM NaCl (A200) and eluted with A200+2 mM biotin 
. Elutions were bound to calmodulin sepharose (GE Healthcare) for 2 hours in A200+2 mM CaCl2
. Beads were washed and eluted with A200+5 mM EGTA. Samples were then TCA precipitated and loaded on 5–20% gradient gels and either silver stained or western blotted.
HeLa cells transduced with the FKBP-Asf1a virus were incubated for 18 hours in the presence of nocodazole (200 ng/ml) or DMSO (0.1%) and Shld1 (1 µM, 
). Shld1 was removed and both suspension and adherent cells were washed twice with PBS followed by addition of fresh media containing nocodazole. At the 18, 24 and 31 hour time-points, suspension and adherent cells were collected, pooled, and divided in two aliquots for Western and FACS analysis. For reversal of the FKBP-Asf1a degradation, cells were incubated with nocodazole in the absence of Shld1 for 18 hours and Shld1 was added for 13 additional hours. Suspension and adherent cells were collected as above. For FACS, cells were resuspended in 200 µl of PBS and fixed with 800 µl of 70% ethanol. RNAse A (100 µg/ml) and propidium iodide (10 µg/ml) were added to the cells before processing on a FACScalibur system and CellQuest software. FACS profiles were displayed using WinMDI 2.9.
FACS analysis and G2 trapping assay for ARID4B overexpression
Twenty-four hours after transduction, puromycin and either DMSO (0.1%) or nocodazole (200 ng/ml) were added to the cells and they were incubated for another 20 hours. Mitotic and cycling cells were harvested by collecting the media and trypsinizing the adherent cells. Cells were pooled and divided in two for Western and FACS analysis (performed as above).
Availability of the plasmids
All the sequences and maps of the plasmids described here are included in the supplementary materials (Table S1
and File S1
). Plasmids will be made available through Addgene (www.addgene.org
). We also provided to Addgene different control vectors to monitor transfection and transduction efficiencies and test for possible toxic effects of the viruses, including some insert-less vectors, or vectors encoding cDNAs or hairpins against GFP or luciferase (see Table S1
for details). Detailed protocols, sequences and maps of our vectors and future vectors will be described at http://www.ericcampeau.com